The cardinal properties of memory CD8 T cells are their longevity, rapid elaboration of effector functions and the ability to proliferate upon re-exposure to the pathogen. We have been addressing fundamental questions about the origin and differentiation of human memory CD8 T cells using the live attenuated yellow fever virus vaccine (YFV-17D) and have made substantial progress during the current cycle of funding. Using in vivo deuterium labeling to mark virus specific CD8 T cells, we have shown that the memory pool originates from CD8 T cells that divided extensively during the first two weeks after infection and is then maintained by quiescent cells that divide less than once a year. Although these long-lived YFV specific memory CD8 T cells did not express effector molecules and had a transcriptional profile similar to nave CD8 T cells, their epigenetic landscape resembled that of virus specific effector CD8 T cells. This open chromatin profile at effector genes was maintained in memory CD8 T cells isolated even a decade after vaccination, indicating that these cells retain an epigenetic fingerprint of their effector history and remain poised to respond rapidly upon re-exposure to the pathogen. These findings have prompted us to ask several new questions about human memory CD8 T cell differentiation. Specifically, what are the transcriptional and epigenetic changes taking place at the single cell level during this effector to memory cell transition? Can we identify fate-permissive and fate-locked YFV specific effector CD8 T cells and follow their differentiation trajectories longitudinally? Is the memory differentiation paradigm that we have defined with our YFV-17D studies generalizable to other acute viral infections? Will memory CD8 T cells after different acute viral infections look the same or will they be different? Does childhood vaccination result in the same memory differentiation program as vaccination of adults? Finally, how are tissue resident memory cells distributed, and how do the phenotype, transcriptional, and epigenetic signatures of these cells compare to the long-lived memory CD8 T cells we have found in the blood. To address these questions the following Specific Aims are proposed: Aim 1. Mapping the differentiation trajectories of virus specific effector CD8 T cells as they transition to long-lived memory. Aim 2. To characterize memory CD8 T cells responses to childhood vaccines and acute viral infections. Aim 3. To analyze virus-specific tissue-